Hematopoietic stem and progenitor cells (HSPC), attracted by the chemokine CXCL12, reside in specific niches in the bone marrow (BM). The long-term objective of our studies is to understand how stem cells egress from the BM into the circulation. This critical process, often referred to as "mobilization", underlies modern clinical stem cell transplantation. Our preliminary studies suggest that signals of the sympathetic nervous system are required for mobilization induced by granulocyte colony-stimulating factor (G-CSF), the most commonly used agent in the clinic. Pharmacological or genetic ablation of adrenergic neurotransmission indicates that norepinephrine (NE) signaling controls G-CSF-induced osteoblast suppression, bone CXCL12 downregulation and HSPC mobilization. In this grant application, we propose to evaluate further the role of adrenergic signals in HSPC mobilization using gain- and loss-of-function mouse models and we will investigate the molecular and cellular basis of collaborating signals. In Specific Aim I, we will evaluate the influence of hyperadrenergic states in HSPC mobilization. We will use strenuous exercise as a physiological model, and mice lacking the NE transporter (Slc6a2-/-) or alpha2A/c adrenergic receptors as genetic models. In Specific Aim II, we will assess the expression of adrenergic receptors in purified osteoblast and osteoclasts and determine their role in HSPC mobilization using knockout animals (loss-of function models). In Specific Aim III, we will characterize G-CSF-induced depletion in bone NE using [3H]NE release and uptake in vivo using radioactive decay methods, and in vitro using SH-SY5Y cells and catecholaminergic neurons derived from embryonic stem cells. In the final Aim, we will test whether the release of active TGF-beta in the stem cell niche contributes to osteoblast suppression and mobilization. We will use in vitro culture systems and Tgfbr2(fi/fl) mice expressing Cre under the MX or Col1a1 promoters, which will delete TGF-beta in an inducible manner or specifically in the osteoblasts, respectively. A function of the sympathetic nervous system in the attraction of stem cells to their niche may explain the conspicuous variability in mobilization efficiencies among normal peripheral blood stem cell donors. In addition, these studies may reveal novel strategies, based on the modulation of the sympathetic outflow to the stem cell niche, to increase the efficiency of HSC harvests for stem cell-based therapeutics.